Stress is known to be an important contributing factor to alcohol abuse and alcoholism, and this is especially relevant to Veterans given their high prevalence of co-occurring alcohol use disorders and stress-related illnesses such as depression and post-traumatic stress disorder (PTSD). However, the interaction between stress and alcohol (ethanol) drinking, as well as mechanisms underlying this interaction in the context of dependence are not well understood. A growing literature has implicated a role for the neurotrophin BDNF (Brain-Derived Neurotrophic Factor) in a number of stress-related psychiatric illnesses (e.g., PTSD) and addiction, including ethanol-related behaviors. During the current funding period, studies were conducted using an established mouse model of ethanol dependence and relapse drinking that led to the discovery of a novel and important neuroadaptive change associated with dependence and related excessive drinking (reduced BDNF activity in the prefrontal cortex). Specifically, repeated cycles of chronic intermittent ethanol (CIE) exposure produced significant reductions in BDNF mRNA/protein expression in dorsomedial prefrontal cortex (dmPFC), and replenishing this deficit via intra-dmPFC BDNF infusion blocked escalated drinking in dependent mice while not altering intake in nondependent mice. Recent pilot studies have shown that stress facilitates this dependence-related escalation of drinking and, like CIE exposure, forced swim stress (FSS) reduces BDNF expression in the dmPFC, as well as in the central amygdala (CeA). Thus, a guiding principle of this proposal is that adaptive changes in BDNF in dmPFC and CeA play a significant role in the ability of stress (FSS) to enhance escalated drinking associated with dependence. Our preliminary data also indicate that both CIE and FSS exposure alter microRNA expression, with evidence suggesting that upregulation of miR-206 in dmPFC may represent a novel mechanism by which BDNF activity is regulated and altered to drive stress enhancement of CIE-induced escalation of drinking. Accordingly, this project will focus on elucidating the role of adaptations in cortical (dmPFC) and subcortical (CeA) BDNF in mediating the interaction between stress and chronic ethanol exposure that leads to excessive levels of drinking associated with dependence. Proposed studies will examine the effects of CIE exposure and FSS exposure, both alone and in combination on time- dependent changes in BDNF mRNA/protein expression in dmPFC and CeA (Aim I). Another set of studies are designed to determine whether viral-mediated overexpression of BDNF in dmPFC or CeA prevents stress (FSS) and CIE induced reductions in BDNF along with their influence on ethanol drinking in the model (Aim II). Finally, a series of studies will determine whether FSS exposure, CIE exposure, and the combination of FSS and CIE exposure alters microRNA (miR-206) regulation of BDNF expression in dmPFC and CeA as a mechanism that contributes to promoting stress enhancement of CIE-induced escalation of drinking (Aim III). These studies will examine effects of FSS, CIE exposure, and combined FSS and CIE exposure on miR-206 expression in dmPFC and CeA in relation to changes in BDNF expression, as well as examine whether CIE- induced and stress enhancement of CIE-induced escalation of drinking is blocked or attenuated by viral- mediated knockdown of miR-206 expression in these target brain regions. Thus, this research proposal will utilize our mouse model of ethanol dependence and relapse drinking to determine mechanisms by which adaptive changes in BDNF activity in dmPFC and CeA contribute to the ability of stress to further augment escalation of drinking associated with ethanol dependence. As such, the proposal addresses a highly significant and clinically important research topic that is of great relevance to Veteran's heath care. The overall goal of the project is to provide new information about potential therapeutic targets for treating ethanol dependence and the exacerbating effects of stress that lead to harmful drinking associated with alcoholism.